内生真菌链格孢菌醋酸乙酯提取物对金黄色葡萄球菌抑菌机制的研究

Antibacterial mechanisms of ethyl acetate extract from endophytic fungi Alternaria alternata against Staphylococcus aureus

目的研究分离自药用植物白毛蛇Humata tyermanni的内生真菌链格孢菌发酵产物醋酸乙酯提取物(B06e)对金黄色葡萄球菌细胞膜完整性和通透性的影响。方法采用二倍稀释法测定B06e对金黄色葡萄球菌的最小抑菌浓度(MIC);测定B06e作用前后金黄色葡萄球菌培养液电导率,206、280 nm处吸光度(A)值的变化并结合流式细胞术研究B06e作用前后细胞膜通透性的变化。利用扫描电子显微镜(SEM)及傅里叶红外变换光谱(FT-IR)研究B06e对细胞膜结构的影响。结果 B06e对金黄色葡萄球菌的MIC为50μg/m L。3×MIC处理组的电导率是对照组的1.06倍;经B06e处理后,反应液的A260和A280值均显著高于对照组;3×MIC处理组的β-半乳糖苷酶活性是对照组的9.43倍;流式细胞仪分析表明3×MIC处理组被碘化丙啶(PI)着染的阳性细胞比例是对照组的47.63倍;SEM和FT-IR分析结果显示,B06e处理后,菌体细胞结构发生了损伤。结论 B06e能增强金黄色葡萄球菌细胞膜的通透性,破坏细胞膜的完整性,造成外源物质的大量流入和细胞质的大量外泄,从而使得菌体细胞内渗透压和代谢调控紊乱,最终导致细菌丧失生长繁殖的能力。

Objective To investigate the effect of ethyl acetate extract（B06 e） from fermentation liquid of an endophytic fungus Alternaria spp. on the cell membrane integrity and the permeability of Staphylococcus aureus. Methods The minimum inhibitory concentration（MIC） of B06 e against S. aureus was measured by double dilution method; The changes of electric conductivity of bacterial culture, A260 and A280 before and after treated by B06 e were analyzed, respectively. Besides, the changes of cell membrane permeability before and after by B06 e was measured by flow cytometry. The effect of B06 e on the cell membrane structure was investigated by scanning electron microscopy（SEM） and Fourier transform infrared spectroscopy（FT-IR）. Results The results showed that MIC value of B06 e against S. aureus was 50 μg/m L. The conductivity of 3 × MIC treatment group was 1.06 times of the value of the control group; after treatment of B06 e, the values of A260 and A280 were significantly higher than those of the control group： The beta-galactosidase activity of 3 × MIC treatment group was 9.43 times more than the value of the control group; Flow cytometry analysis showed that 3 × MIC treatment group by propidium iodide（PI） staining of positive cells was 47.63 times more than the control group; SEM and FT-IR analysis showed that the structure of bacterial cell changed after B06 e treatment. Conclusion B06 e can kill S. aureus cell by increasing the permeability of its cell membrane and destroy cell membrane integrity.